1. Field of the Invention
The present invention relates to a novel recombinant yeast substrain, especially to a recombinant yeast substrain suitable for the production of ethanol by fermentation under 42° C. with high efficiency. The recombinant yeast substrain was constructed by replacing the genomic regulatory region for yeast gene hsp104 using linear DNA transformation, so as to alter the behavior of Hsp104 protein under stress and to facilitate the production of alcohol by high-temperature fermentation.
2. Description of the Prior Art
Biomass ethanol is a biomass fuel that generates bio-energy and is obtained by conversion of biomass. Said biomass may be molasses or plants such as maize, wheat or potatoes that allow production of biomass ethanol through the processes of fermentation and distillation. Biomass ethanol is known as a feasible way to reduce dependence on fossil fuels.
Materials for making biomass ethanol are roughly classified into three categories:
1) sugar materials derived from monosaccharide-abundant sugar corps such as sugar cane or sorgo;
2) starch materials derived from wheat or corn; and
3) cellulosic materials derived from agricultural wastes.
Materials of the first and second categories primarily come from food corps. Using such material leads to the zero-sum competition between food and energy applications that are both based on food corps. Materials of the third category, though raise no competition between food and energy applications, require a high production cost yet to be overcome.
The method for making ethanol from the cellulosic materials of the third category may be largely divided into four stages:
1) pretreatment with weak acid, weak base or ammonia-gas explosions to separate cellulose or hemicellulose from the complex comprising binding lignin, so as to facilitate the chemical or biological processing in following stages.
2) degeneration or hydrolysis for obtaining free sugars;
3) fermentation of mixed hexose and pentose to produce ethanol; and
4) collection and distillation of the product for obtaining biomass ethanol.
One of the means for lowering the production cost is to synchronize the degeneration/hydrolysis stage and the fermentation stage.
However, the operation temperature for the cellulose enzyme employed in the degeneration/hydrolysis stage is approximately 45-60° C., which is higher than the fermentation temperatures of most industrial brewing yeasts (Saccharomyces cerevisiae). Should a yeast with high-temperature tolerance capable of working under the high temperature of the degeneration/hydrolysis stage be cultivated, such a thermotolerant yeast would synchronously proceed the tasks of the degeneration/hydrolysis stage and the fermentation stage, so as to lower the production cost of biomass ethanol.
Only a few of antecedent technologies are relevant to thermotolerant yeast as below listed works:
Yamada et al. (US patent application publication number: US 2010/0062506), with screening media containing high concentration of sugar and alcohol thermotolerant, have isolated yeast Kluyveromyces marxianus capable of producing ethanol by fermentation of sugar cane juice and peaking best productivity as high as 1.51 g of ethanol per liter per hour at 40° C.
Forrester et al. (US patent application publication number: US 2011/0033907) have isolated Saccharomyces cerevisiae strains YE1358 and YE1615. At 37° C., fermentation of 250 g/L maize flour and 2 mM CaCl2 with YE1615 gives approximately 130 g/L ethanol.
Abbas et al. (US patent application publication number: US 2009/0155872) have constructed a plasmid comprising 1) H. polymorpha (P. angusta) glyceraldehyde-3 phosphate dehydrogenase (GADPH) promoter-heat shock protein 104 (hsp104) gene and 2) GADPH promoter-xylulokinase. Transforming H. polymorpha with the plasmid suppresses the activity of acid trehalase (ATH1) and raises the capability of H. polymorpha to ferment 12% xylose for producing ethanol.
Edgardo et al. (Edgardo et al. 2008, Enzym. Microb. Tech. 43, 120-123) have screened Saccharomyces cerevisiae at 35-45° C. to isolate strains capable of growing and fermenting glucose at 42° C. However, the fermentation efficiency of the strain is 75% lower than the theoretical value in a solution of 50 g/L glucose concentration at 40° C., and is 25% lower at 52° C.
Benjaphokee et al. (Benjaphokee et al. 2012, N. Biotechnol. 29, 379-386), by crossing the spores of thermotolerat Saccharomyces cerevisiae HB8-3A and ethanol-productive Saccharomyces cerevisiae TISTR5606, have obtained a strain, TJ14, capable of fermenting glucose at 41° C. in a pH 3.5 solution of glucose concentration 100 g/L with a peak fermentation efficiency as high as 90%.
Lindquist and Kim have identified that a molecular chaperone in yeast, the molecular chaperone Hsp104, is capable of separating gathered proteins and refold the same, which raises the thermotolerance of the yeast (Bösl, et al. 2006, J. Struct. Biol. 156, 139-148). Hsp104 and Hsp70/40 extract polypeptide chains out from an aggregated protein complex and facilitate the refolding of the same (Lee et al. 2003, Cell 115, 229-240; Waghmare et al. 2003, Biotechniques 34, 1024-1028; Storici et al. 1999, Yeast 15, 271-283; Weibezahn et al. 2005, Biol. Chem. 386, 739-744). Heat shock protein and other molecular chaperones are indispensable for cellular stasis of a cell. In normal circumstances, it is vital that these chaperones are sufficiently expressed. When under environmental stress, misfolded proteins accumulate and disrupt cellular physiological conditions. Misfolded proteins may further induce generation of chaperones, which help restore and maintain normal cellular physiological conditions. Comparing with other chaperones, Hsp104 is insignificant under normal conditions and thus its low concentration. However, under fatal environmental stress, the concentration of Hsp104 acutely rises in a short period of time to restore the activities of the disabled proteins accumulated in the cell. In Saccharomyces cerevisiae, Hsp104, though expression level of which raises responsive to a stress, the expression level lowers in a few hours, which fails to allow Saccharomyces cerevisiae to survive at high temperature for a significantly long period of time and thus makes Saccharomyces cerevisiae unsuitable for synchronized hydrolysis and fermentation.
Altered expression of recombinant genes to enhance tolerance of yeasts to stress is practicable. However, few available recombination tags tend to be insufficient. In order to reuse the recombination tags that are few in number, the FLP/FRT recognition-site-specific recombination system is employed. The natural flippase recombinase gene, flp, is derived from the 2 μm plasmid of Saccharomyces cerevisiae; FRT stands for “Flippase recombinase Recognition Target.” The FRT comprises 34 base pairs (bp/bps): SEQ ID NO: 12, and is divided into two regions. The first consecutive 13 bps and last 13 consecutive bps belong to a complementary region, which is the FLP recognition site. The central 8 bps are named as the core region, which is asymmetric. FLP recognizes two identical FRT in the same direction and flip-out the gene flanked by the two FRT to accomplish specific gene recombination. The possibility of the occurrence of recombination based on two non-identical FRTs is extreme low (Storici et al. 1999, Yeast 15, 271-283).
Due to the lack of thermotolerant Saccharomyces cerevisiae having glucose fermentation efficiency higher than 95% and method for making same, it is apparent that there is a present need for such means to lower the production cost of cellulosic biomass ethanol.
To overcome the shortcomings that the prior art fails to provide a thermotolerant yeast and fails to lower the production cost for biomass ethanol, the present invention provides a thermotolerant yeast with a substitute heat shock protein 104 promoter to mitigate or obviate the aforementioned problems.